Compositions and methods for modulating hemostasis

ABSTRACT

Factor Xa variants and methods of use thereof are disclosed.

This application is a divisional application of U.S. patent applicationSer. No. 13/726,187 filed Dec. 23, 2012, which is a divisionalapplication of U.S. patent application Ser. No. 12/093,783, filed Jul.16, 2008 which is a National Phase application of PCT/US06/60927 filedNov. 15, 2006, which in turn claims priority to U.S. ProvisionalApplication 60/736,680 filed Nov. 15, 2005, the entire contents of eachbeing incorporated herein by reference as though set forth in full.

This invention was made with government support under Grant No. PO1HL-74124-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and hematology.More specifically, the invention provides novel coagulation Factor X/Xaagents and methods of using the same to modulate the coagulation cascadein patients in need thereof.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

The enzymes of coagulation are trypsin-like enzymes that belong to theS1 peptidase family of proteases that bear a chymotrypsin-like fold. Thecoagulation proteases contain catalytic domains that are highlyhomologous to each other and to the ancestral serine proteases ofdigestion. The structural homology/identity is so great (>70%) thatresidues in the catalytic domains of the coagulation enzymes arenumbered according to the corresponding residues in chymotrypsinogen.

The coagulation enzymes circulate in blood as inactive precursors,zymogens, that require proteolytic cleavage for activation. The zymogenspossess ˜10,000-fold or less proteolytic activity when compared to theserine proteases produced following activation. Initiation ofcoagulation at the site of vascular damage leads to a series ofreactions in which a zymogen is converted to a protease through specificproteolytic cleavage and forms the enzyme for the successive reaction.This culminates in blood cell activation and the conversion of solublefibrinogen to insoluble fibrin and hence the formation of the clot.Excess proteases are removed by reaction with circulating proteaseinhibitors that act as “suicide” substrates or those that recognize theactive enzymes. Thus, proteolytic activation of the coagulation zymogensis a key regulatory feature of the coagulation cascade.

Although some of the coagulation zymogens are cleaved at two or moresites in their respective activation reactions, formation of theprotease requires cleavage at a single site. Cleavage at this site andits structural consequences are considered in the most facile way usingthe homologous numbering system based on chymotrypsinogen and theextensive structural work done with trypsinogen and trypsin. Theconversion of the zymogen to serine protease requires cleavage followingArg¹⁵ (typically the bond between Arg¹⁵ and Ile¹⁶) which typicallyremoves an activation peptide and exposes a new N-terminus in thecatalytic domain beginning with Ile¹⁶. One example is the conversion offactor X to factor Xa (see FIGS. 1 and 2). In trypsin and factor Xa, thenew N-terminal sequence begins with Ile¹⁶-Val¹⁷-Gly¹⁸-Gly¹⁹ (SEQ ID NO:4). For other clotting enzymes, the new N-terminal sequence is avariation on the same theme. The N-terminal sequence then folds backinto the catalytic domain and inserts into the N-terminal binding cleftin a sequence-specific manner which is referred to as “molecularsexuality”. See FIG. 2. Accordingly, variants with alternate N-terminalsequences are not likely to undergo molecular sexuality in a comparableway. N-terminal insertion leads to the formation of a salt bridgebetween the α-NH₂ group of Ile¹⁶ and Asp¹⁹⁴ in the interior of thecatalytic domain. Salt bridge formation is associated with numerouschanges in catalytic domain structure including: rearrangements of theso-called activation domains, shown in FIG. 3; formation of the oxyanionhole required for catalysis and the formation of a substrate bindingsite. These changes lead to the maturation of the active serineprotease. The key contribution of sequence-specific interactions of thenew N-terminus through molecular sexuality and salt bridge formation tothe maturation of the active protease are evident from the followingfacts: bacterial proteases that do not require cleavage for activationutilize another side-chain within the catalytic domain to salt bridgewith Asp¹⁹⁴; trypsinogen can be activated to a proteinase-likeconformation without cleavage but with extremely high concentrations ofan Ile-Val dipeptide that inserts into the cleft, albeit veryinefficiently; the Val-Ile dipeptide and other variants are far lesseffective; additionally, there are two examples of bacterial proteinsthat activate coagulation zymogens in the absence of cleavage bysubverting the activation mechanism via provision of their ownN-terminus that inserts into the N-terminal binding cleft.

The structural changes outlined above provide a molecular explanationfor the conversion of a precursor zymogen to an active serine protease.However, unlike trypsin which is fully active following cleavage atArg¹⁵, many of the coagulation enzymes act very poorly on their proteinsubstrates. Even though they generally possess fully functional activesites and can cleave small peptidyl substrates, efficient cleavage ofthe biological substrate often requires a cofactor protein (FIG. 2). Inthese cases, the cofactor proteins increase the rate of proteinsubstrate cleavage by several thousand fold. Although the mechanism bywhich the cofactor proteins function remains to be resolved, they areunlikely to function by making the protease more enzyme-like andtherefore more efficient. A key point is that, with one exception, thecofactors selectively bind the protease and not the correspondingzymogen. For example, factor Xa binds with high affinity tomembrane-bound FVa, whereas the zymogen factor X does not bind FVa.

Depending on the state of the patient it may be desirable to developaltered coagulation cascade proteins which possess enhanced or reducedcoagulation function. It is an object of the invention to provide suchproteins for use as therapeutics.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided for influencing regulatory sites in the FX zymogen→proteasetransition pathway thereby driving production of a more “zymogen-like”FXa species. The compositions and methods of the invention are effectiveto modulate hemostasis in patients in need thereof.

In one embodiment, a variant Factor X/Factor Xa zymogen/protease whichmodulates hemostasis is provided. Preferably, the variant zymogenprotease is encoded by SEQ ID NO: 2, wherein nucleotides 1684-1695 ofSEQ ID NO: 2 can be any amino acid with the proviso that nucleotides1684-1886 do not encode Val or Ala. More preferably, the variantzymogen/protease contains at least one modification in SEQ ID NO: 1selected from the group consisting of a) Ile at position 16 is Leu, Phe,Asp or Gly; b) Val at position 17 is Leu, Ala, or Gly and c) Asp atposition 194 is Asn or Glu. Nucleic acids encoding the variantzymogen/proteases of the invention are also disclosed as are methods ofuse thereof. Such nucleotides may optionally encode an intracellularPACE/furin cleavage site.

In yet another embodiment, a nucleic acid having the sequence of SEQ IDNO: 2, wherein the nucleotides at positions 1684-1695 encode the aminoacids selected from the group consisting of Leu-Val-Gly, Gly-Val-Gly,Ile-Ala-Gly, Phe-Val-Gly and Ile-Gly-Gly, said nucleic acid optionallycomprising nucleotides at position 2233-2235 which encode an amino acidselected from the group consisting of Asn or Glu.

A pharmaceutical composition comprising the Factor Xa variant of theinvention in a biologically compatible carrier is also provided. Anotherpreferred aspect of the invention includes methods for the treatment ofa hemostasis related disorder in a patient in need thereof comprisingadministration of a therapeutically effective amount of the variantFactor X/Xa zymogen/protease containing pharmaceutical compositionsdescribed herein. Such methods should have efficacy in the treatment ofdisorders where a pro-coagulant is needed and include, withoutlimitation, hemophilia A and B, hemophilia A and B associated withinhibitory antibodies, coagulation factor deficiency, vitamin K epoxidereductase C1 deficiency, gamma-carboxylase deficiency, bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation treatment disorders, Bernard Soulier syndrome,Glanzman thromblastemia, and storage pool deficiency.

Certain zymogen/protease variants may be useful in the treatment ofdisorders where anti-coagulation is desired. Such disorders include,without limitation, thrombosis, thrombocytopenia, stroke, andcoagulopathy.

Another aspect of the invention, includes host cells expressing thevariant zymogen/proteases of the invention in order to produce largequantities thereof. Methods for isolating and purifying the zymogenprotease variants are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Processing of Factor X. Factor X is synthesized with a signalsequence and propeptide which are removed prior to its secretion. FactorX is a zymogen and has no enzymatic activity. FX is converted to factorXa following cleavage at Arg15-Ile 16 bond releasing an activationpeptide (AP). The ANSF sequence is SEQ ID NO: 5 and the TLER sequence isSEQ ID NO: 6.

FIG. 2. Zymogen to protease conversion. The zymogen to proteasetransition for factor X and assembly of factor Xa into prothrombinase(FXa, FVa, phospholipid and calcium ions). This enzyme convertsprothrombin (II) to thrombin (IIa). The IVGG sequence is SEQ ID NO: 4.

FIG. 3. The X-ray structure of FXa. The catalytic domain of FXa in thestandard orientation. Structural regions are noted along with importantresidues. Taken from Brandstetter et al. (1996) J. Biol. Chem.271:29988-29992.

FIG. 4. SDS-PAGE analysis of FX/Xa variants. 4-12% SDS-PAGE gels wererun under either non-reducing or reducing conditions and then stainedwith Coomassie Blue.

FIGS. 5A-5D. Amino acid (SEQ ID NO: 1) and nucleic acid (SEQ ID NO: 2)sequences of Factor Xa. The sites and amino acid positions for desiredmodifications in SEQ ID NO: 1 are shown in bold.

FIG. 6. Factor Xa activity in hemophilia B plasma. Wild-type FXa orFXaI16L (2 nM) were added to hemophilia B plasma and at select timeintervals the samples were diluted (0.1 nM) and assayed in an aPTTclotting assay.

FIG. 7. Correction of the aPTT. Factor Xa-I16L (200 μg/kg; n=7 mice) orPBS (n=4 mice) were injected into hemophilia B mice (C57BL/6) via thetail vein. At 5 and 30 min post-injection, blood was collected and anaPTT assay was performed. The red dotted line represents the aPTT valueof normal C57Bl/6 animals.

FIG. 8. Hemostatic assessment following tail-clip assay in hemophilia Bmice. Blood loss is measured by the hemoglobin content of the salinesolution by A525 post-injury. The number of mice (Balb c) are; wild-type(n=7); HB-PBS (n=6); and HB-FXaI16L (n=7).

DETAILED DESCRIPTION OF THE INVENTION

Proteolysis is an essential aspect of blood coagulation and underliesmany of the mechanisms regulating normal hemostasis. Procofactors andzymogens cannot participate to any significant degree in theirrespective macromolecular enzymatic complexes. This indicates thatproteolytic activation must result in appropriate structural changesthat lead to the expression of sites which impart enzyme, substrate andcofactor binding capabilities. While procofactor and zymogen activationhas been intensively studied, the relationship between proteolysis andthe expression of binding sites which impart function is incompletelyunderstood. The present invention provides model compositions andsystems which elucidate the molecular mechanisms underlying theexpression of macromolecular binding interactions that accompanytransitions from the zymogen state.

Factor X (FX)¹ is a vitamin K-dependent two-chain glycoprotein whichplays a central role in blood coagulation (FIG. 1). This serine proteasezymogen is a substrate for both the extrinsic (tissue factor/FVIIa) andintrinsic (FVIIIa/FIXa) tenase enzyme complexes which cleave theArg¹⁵-Ile¹⁶ scissile bond in FX releasing a 52-amino acid activationpeptide generating FXa. Factor Xa is the protease responsible for theconversion of prothrombin to thrombin (FIG. 2). Although factor Xa is afully competent protease and possesses the catalytic machinery for thecleavage of prothrombin, it is a profoundly poor catalyst for thisreaction. Its tight binding interaction with the cofactor, factor Va, ona membrane surface profoundly increases the rate of thrombin formationwithout substantially affecting other reactions catalyzed by factor Xa.Changes to the N-terminal sequence (Ile-Val-Gly) following the Arg15cleavage site that lead to suboptimal molecular sexuality are expectedto yield a “zymogen-like” Xa derivative that has impaired, or even zero,proteolytic activity. These derivatives are not expected to besusceptible to inhibition by plasma protease inhibitors such asAntithrombin III and are not expected to interfere with the initiationof coagulation following vascular damage because they are not expectedto bind TFPI very well. Factor Xa binds factor Va tightly while thezymogen factor X does not. Thus, zymogen-like forms of factor Xa areexpected to bind Va more weakly but be completely rescued atsufficiently high cofactor concentrations and catalyze thrombinformation efficiently. Zymogen-like forms of factor Xa with theseproperties are expected to act as long-lived proteases in circulationthat are otherwise dead but retain the ability to catalyze thrombinformation upon binding to factor Va. They have the potential to serve astherapeutic procoagulants that bypass deficiencies in other clottingfactors in the cascade, without the deleterious effects associated withinfusion of fully functional wild type FXa.

I. Definitions

Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specification andclaims.

The phrase “variant zymogen/protease” refers to a modified FX zymogen orFXa protease which has been genetically altered such that its proteaseactivity when converted to FXa is reduced or “zymogen-like” in theabsence of specific cofactors (e.g., the binding affinity for the activesite is lower than that observed in the wild type molecule. Notably,this affinity/activity is restored in the presence of the properco-factors which include, without limitation factor Va. Preferred sitesfor amino acid alterations in the parent FX molecule includesubstitution of the isoleucine at position 16, substitution of thevaline at position 17 and substitution of the aspartic acid at position194, with the proviso that the amino acid at position 16 is not valineor alanine.

The phrase “hemostasis related disorder” refers to bleeding disorderssuch as hemophilia A and B, hemophilia A and B patients with inhibitoryantibodies, deficiencies in coagulation Factors, VII, IX and X, XI, V,XII, II, von Willebrand factor, combined FV/FVIII deficiency, vitamin Kepoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation associated with heparin, low molecular weightheparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e.FXa inhibitors); and platelet disorders such as, Bernard Souliersyndrome, Glanzman thromblastemia, and storage pool deficiency.

A hemostasis related disorder can also include bleeding related tothromboic disorders such as deep venous thrombosis, thrombosisassociated with cardiovascular disease states or malignancies,thrombosis resulting from in-dwelling catheters or other invasivesurgical procedures and thrombosis associated with autoimmune diseasessuch as lupus. The zymogen/protease variants could also providenecessary hemostasis for patients with disseminated intravascularcoagulation or consumptive coagulopathies arising from a variety ofdisease states.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it originates. For example,the “isolated nucleic acid” may comprise a DNA or cDNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into theDNA of a prokaryote or eukaryote.

With respect to RNA molecules of the invention, the term “isolatednucleic acid” primarily refers to an RNA molecule encoded by an isolatedDNA molecule as defined above. Alternatively, the term may refer to anRNA molecule that has been sufficiently separated from RNA moleculeswith which it would be associated in its natural state (i.e., in cellsor tissues), such that it exists in a “substantially pure” form (theterm “substantially pure” is defined below).

With respect to protein, the term “isolated protein” or “isolated andpurified protein” is sometimes used herein. This term refers primarilyto a protein produced by expression of an isolated nucleic acid moleculeof the invention. Alternatively, this term may refer to a protein whichhas been sufficiently separated from other proteins with which it wouldnaturally be associated, so as to exist in “substantially pure” form.

The term “promoter region” refers to the transcriptional regulatoryregions of a gene, which may be found at the 5′ or 3′ side of the codingregion, or within the coding region, or within introns.

The term “vector” refers to a small carrier DNA molecule into which aDNA sequence can be inserted for introduction into a host cell where itwill be replicated. An “expression vector” is a specialized vector thatcontains a gene or nucleic acid sequence with the necessary regulatoryregions needed for expression in a host cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of a coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector. This definition is alsosometimes applied to the arrangement of nucleic acid sequences of afirst and a second nucleic acid molecule wherein a hybrid nucleic acidmolecule is generated.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-99% by weight,of the compound of interest. Purity is measured by methods appropriatefor the compound of interest (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

The phrase “consisting essentially of” when referring to a particularnucleotide sequence or amino acid sequence means a sequence having theproperties of a given SEQ ID NO. For example, when used in reference toan amino acid sequence, the phrase includes the sequence per se andmolecular modifications that would not affect the basic and novelcharacteristics of the sequence.

The term “oligonucleotide,” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo- or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application for which the oligonucleotideis used.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and method of use. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides.

The probes herein are selected to be “substantially” complementary todifferent strands of a particular target nucleic acid sequence. Thismeans that the probes must be sufficiently complementary so as to beable to “specifically hybridize” or anneal with their respective targetstrands under a set of pre-determined conditions. Therefore, the probesequence need not reflect the exact complementary sequence of thetarget. For example, a non-complementary nucleotide fragment may beattached to the 5′ or 3′ end of the probe, with the remainder of theprobe sequence being complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to act functionally as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product.

The primer may vary in length depending on the particular conditions andrequirements of the application. For example, in diagnosticapplications, the oligonucleotide primer is typically 15-25 or morenucleotides in length. The primer must be of sufficient complementarityto the desired template to prime the synthesis of the desired extensionproduct, that is, to be able to anneal with the desired template strandin a manner sufficient to provide the 3′ hydroxyl moiety of the primerin appropriate juxtaposition for use in the initiation of synthesis by apolymerase or similar enzyme. It is not required that the primersequence represent an exact complement of the desired template. Forexample, a non-complementary nucleotide sequence may be attached to the5′ end of an otherwise complementary primer. Alternatively,non-complementary bases may be interspersed within the oligonucleotideprimer sequence, provided that the primer sequence has sufficientcomplementarity with the sequence of the desired template strand tofunctionally provide a template-primer complex for the synthesis of theextension product.

The term “percent identical” is used herein with reference tocomparisons among nucleic acid or amino acid sequences. Nucleic acid andamino acid sequences are often compared using computer programs thatalign sequences of nucleic or amino acids thus defining the differencesbetween the two. For purposes of this invention comparisons of nucleicacid sequences are performed using the GCG Wisconsin Package version9.1, available from the Genetics Computer Group in Madison, Wis. Forconvenience, the default parameters (gap creation penalty=12, gapextension penalty=4) specified by that program are intended for useherein to compare sequence identity. Alternately, the Blastn 2.0 programprovided by the National Center for Biotechnology Information (found onthe world wide web at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, JMol Biol 215:403-410) using a gapped alignment with default parameters,may be used to determine the level of identity and similarity betweennucleic acid sequences and amino acid sequences.

II. Preparation of Variant Zymogen-Protease Encoding Nucleic AcidMolecules and Polypeptides

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the variant zymogen/proteases of theinvention may be prepared by using recombinant DNA technology methods.The availability of nucleotide sequence information enables preparationof isolated nucleic acid molecules of the invention by a variety ofmeans. For example, nucleic acid sequences encoding a zymogen/proteasepolypeptide may be isolated from appropriate biological sources usingstandard protocols well known in the art.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in a plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell. Alternatively, the nucleic acids may be maintained invector suitable for expression in mammalian cells. In cases wherepost-translational modification affects zymogen/protease function (e.g.,Factor Xa), it is preferable to express the molecule in mammalian cells.

In one embodiment, the nucleic acids encoding the factor X zymogenvariants may be further modified via insertion of an intracellularproteolytic cleavage site. In order to express “activated” zymogen-likeFXa variants in mammalian cells, an intracellular proteolytic cleavagesite can be inserted between positions Arg15 and 16 in the variant FXzymogen. Such cleavage sites include: Arg-Lys-Arg orArg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO: 3). These cleavage sites areefficiently recognized by proteases (PACE/furin-like enzymes) within thecell and are removed. This results in a processed variant FX(a) in whichthe heavy chain on the molecule begins now begins at position 16.Introduction of this cleavage site at said position will allow for theintracellular conversion of FX to FXa.

In another embodiment, the entire 52 amino acid activation peptide canbe removed and the intracellular protease cleavage site can beintroduced in its place which will result in variant FXa.

Ultimately these types of modifications allow for secretion of the“active” processed form of variant FX from a cell that expresses themodified variant FX. Secretion of the cleaved factor obviates a need forproteolytic cleavage during blood clotting or following the isolation ofthe protein.

Variant zymogen/protease-encoding nucleic acid molecules of theinvention include cDNA, genomic DNA, RNA, and fragments thereof whichmay be single- or double-stranded. Thus, this invention providesoligonucleotides (sense or antisense strands of DNA or RNA) havingsequences capable of hybridizing with at least one sequence of a nucleicacid molecule of the present invention. Such oligonucleotides are usefulas probes for detecting zymogen/protease expression.

B. Proteins

A full-length or variant zymogen/protease polypeptide of the presentinvention may be prepared in a variety of ways, according to knownmethods. The protein may be purified from appropriate sources, e.g.,transformed bacterial or animal cultured cells or tissues which expresszymogen/protease, by immunoaffinity purification. However, this is not apreferred method due to the low amount of protein likely to be presentin a given cell type at any time.

The availability of nucleic acid molecules encoding a variantzymogen/protease polypeptide enables production of zymogen/proteaseusing in vitro expression methods known in the art. For example, a cDNAor gene may be cloned into an appropriate in vitro transcription vector,such as pSP64 or pSP65 for in vitro transcription, followed by cell-freetranslation in a suitable cell-free translation system, such as wheatgerm or rabbit reticulocyte lysates. In vitro transcription andtranslation systems are commercially available, e.g., from PromegaBiotech, Madison, Wis. or BRL, Rockville, Md.

Alternatively, according to a preferred embodiment, larger quantities ofzymogen/protease may be produced by expression in a suitable prokaryoticor eukaryotic expression system. For example, part or all of a DNAmolecule encoding variant Factor Xa for example, may be inserted into aplasmid vector adapted for expression in a bacterial cell, such as E.coli or a mammalian cell such as CHO or Hela cells. Alternatively, in apreferred embodiment, tagged fusion proteins comprising zymogen/proteasecan be generated. Such zymogen/protease-tagged fusion proteins areencoded by part or all of a DNA molecule, ligated in the correct codonreading frame to a nucleotide sequence encoding a portion or all of adesired polypeptide tag which is inserted into a plasmid vector adaptedfor expression in a bacterial cell, such as E. coli or a eukaryoticcell, such as, but not limited to, yeast and mammalian cells. Vectorssuch as those described above comprise the regulatory elements necessaryfor expression of the DNA in the host cell positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include, but are not limited to,promoter sequences, transcription initiation sequences, and enhancersequences.

Variant zymogen/protease proteins, produced by gene expression in arecombinant prokaryotic or eukaryotic system may be purified accordingto methods known in the art. In a preferred embodiment, a commerciallyavailable expression/secretion system can be used, whereby therecombinant protein is expressed and thereafter secreted from the hostcell, to be easily purified from the surrounding medium. Ifexpression/secretion vectors are not used, an alternative approachinvolves purifying the recombinant protein by affinity separation, suchas by immunological interaction with antibodies that bind specificallyto the recombinant protein or nickel columns for isolation ofrecombinant proteins tagged with 6-8 histidine residues at theirN-terminus or C-terminus. Alternative tags may comprise the FLAGepitope, GST or the hemagglutinin epitope. Such methods are commonlyused by skilled practitioners.

Zymogen/protease proteins, prepared by the aforementioned methods, maybe analyzed according to standard procedures. For example, such proteinsmay be subjected to amino acid sequence analysis, according to knownmethods.

As discussed above, a convenient way of producing a polypeptideaccording to the present invention is to express nucleic acid encodingit, by use of the nucleic acid in an expression system. A variety ofexpression systems of utility for the methods of the present inventionare well known to those of skill in the art.

Accordingly, the present invention also encompasses a method of making apolypeptide (as disclosed), the method including expression from nucleicacid encoding the polypeptide (generally nucleic acid). This mayconveniently be achieved by culturing a host cell, containing such avector, under appropriate conditions which cause or allow production ofthe polypeptide. Polypeptides may also be produced in in vitro systems,such as in reticulocyte lysates.

III. Uses of Zymogen/protease Proteins and Zymogen/Protease-EncodingNucleic Acids

Variant zymogen/protease nucleic acids encoding polypeptides havingaltered protease activities may be used according to this invention, forexample, as therapeutic and/or prophylactic agents (protein or nucleicacid) which modulate the blood coagulation cascade. The presentinventors have discovered that factor X/Xa zymogen/protease moleculescan increase coagulation and provide effective hemostasis.

A. Variant Zymogen/Protease Polypeptides

In a preferred embodiment of the present invention, variantzymogen/protease polypeptides may be administered to a patient viainfusion in a biologically compatible carrier, preferably viaintravenous injection. The variant zymogen/proteases of the inventionmay optionally be encapsulated into liposomes or mixed with otherphospholipids or micelles to increase stability of the molecule.Zymogen/protease may be administered alone or in combination with otheragents known to modulate hemostasis (e.g., Factor V, Factor Va orderivatives thereof). An appropriate composition in which to deliverzymogen/protease polypeptides may be determined by a medicalpractitioner upon consideration of a variety of physiological variables,including, but not limited to, the patient's condition and hemodynamicstate. A variety of compositions well suited for different applicationsand routes of administration are well known in the art and are describedhereinbelow.

The preparation containing the purified factor X/Xa analog contains aphysiologically acceptable matrix and is preferably formulated as apharmaceutical preparation. The preparation can be formulated usingsubstantially known prior art methods, it can be mixed with a buffercontaining salts, such as NaCl, CaCl₂, and amino acids, such as glycineand/or lysine, and in a pH range from 6 to 8. Until needed, the purifiedpreparation containing the factor X/Xa analog can be stored in the formof a finished solution or in lyophilized or deep-frozen form. Preferablythe preparation is stored in lyophilized form and is dissolved into avisually clear solution using an appropriate reconstitution solution.

Alternatively, the preparation according to the present invention canalso be made available as a liquid preparation or as a liquid that isdeep-frozen.

The preparation according to the present invention is especially stable,i.e., it can be allowed to stand in dissolved form for a prolonged timeprior to application.

The preparation according to the present invention which contains afactor X analog in combination with factor XIa or a derivative thereofwhich is able to activate the factor X analog into factor Xa or thefactor Xa analog can be made available in the form of a combinationpreparation comprising a container that holds factor XIa which isimmobilized on a matrix, potentially in the form of a miniature columnor a syringe complemented with a protease, and a container containingthe pharmaceutical preparation with the factor X analog. To activate thefactor X analog, the factor X analog-containing solution, for example,can be pressed over the immobilized protease. During storage of thepreparation, the factor X analog-containing solution is preferablyspatially separated from the protease. The preparation according to thepresent invention can be stored in the same container as the protease,but the components are spatially separated by an impermeable partitionwhich can be easily removed before administration of the preparation.The solutions can also be stored in separate containers and be broughtinto contact with each other only shortly prior to administration.

The factor X analog can be activated into factor Xa shortly beforeimmediate use, i.e., prior to the administration to the patient. Theactivation can be carried out by bringing a factor X analog into contactwith an immobilized protease or by mixing solutions containing aprotease, on the one hand, and the factor X analog, on the other hand.Thus, it is possible to separately maintain the two components insolution and to mix them by means of a suitable infusion device in whichthe components come into contact with each other as they pass throughthe device and thereby to cause an activation into factor Xa or into thefactor Xa analog. The patient thus receives a mixture of factor Xa and,in addition, a serine protease which is responsible for the activation.In this context, it is especially important to pay close attention tothe dosage since the additional administration of a serine protease alsoactivates endogenous factor X, which may shorten the coagulation time.

The preparation according to the present invention can be made availableas a pharmaceutical preparation with factor Xa activity in the form of aone-component preparation or in combination with other factors in theform of a multi-component preparation.

Prior to processing the purified protein into a pharmaceuticalpreparation, the purified protein is subjected to the conventionalquality controls and fashioned into a therapeutic form of presentation.In particular, during the recombinant manufacture, the purifiedpreparation is tested for the absence of cellular nucleic acids as wellas nucleic acids that are derived from the expression vector, preferablyusing a method, such as is described in EP 0 714 987.

Another feature of this invention relates to making available apreparation which contains a factor Xa analog with a high stability andstructural integrity and which, in particular, is free from inactivefactor X/Xa analog intermediates and autoproteolytic degradationproducts and which can be produced by activating a factor X analog ofthe type described above and by formulating it into an appropriatepreparation.

The pharmaceutical preparation may contain dosages of between 10-1000μg/kg, more preferably between about 10-250 μg/kg and most preferablybetween 10 and 75 μg/kg, with 40 μg/kg of the variant factor Xpolypeptide being particularly preferred. Patients may be treatedimmediately upon presentation at the clinic with a bleed. Alternatively,patients may receive a bolus infusion every one to three hours, or ifsufficient improvement is observed, a once daily infusion of the variantfactor Xa described herein.

B. Zymogen/Protease-Encoding Nucleic Acids

Zymogen/protease-encoding nucleic acids may be used for a variety ofpurposes in accordance with the present invention. In a preferredembodiment of the invention, a nucleic acid delivery vehicle (i.e., anexpression vector) for modulating blood coagulation is provided whereinthe expression vector comprises a nucleic acid sequence coding for avariant zymogen/protease polypeptide, or a functional fragment thereofas described herein. Administration of zymogen/protease-encodingexpression vectors to a patient results in the expression ofzymogen/protease polypeptide which serves to alter the coagulationcascade. In accordance with the present invention, an zymogen/proteaseencoding nucleic acid sequence may encode an zymogen/proteasepolypeptide as described herein whose expression increases hemostasis.In a preferred embodiment, a zymogen/protease nucleic acid sequenceencodes a human Factor Xa polypeptide variant.

Expression vectors comprising variant X/Xa zymogen/protease nucleic acidsequences may be administered alone, or in combination with othermolecules useful for modulating hemostasis. According to the presentinvention, the expression vectors or combination of therapeutic agentsmay be administered to the patient alone or in a pharmaceuticallyacceptable or biologically compatible compositions.

In a preferred embodiment of the invention, the expression vectorcomprising nucleic acid sequences encoding the variant zymogen/proteasevariants is a viral vector. Viral vectors which may be used in thepresent invention include, but are not limited to, adenoviral vectors(with or without tissue specific promoters/enhancers), adeno-associatedvirus (AAV) vectors of multiple serotypes (e.g., AAV-2, AAV-5, AAV-7,and AAV-8) and hybrid AAV vectors, lentivirus vectors and pseudo-typedlentivirus vectors [e.g., Ebola virus, vesicular stomatitis virus (VSV),and feline immunodeficiency virus (FIV)], herpes simplex virus vectors,vaccinia virus vectors, and retroviral vectors.

In a preferred embodiment of the present invention, methods are providedfor the administration of a viral vector comprising nucleic acidsequences encoding a variant zymogen/protease, or a functional fragmentthereof. Adenoviral vectors of utility in the methods of the presentinvention preferably include at least the essential parts of adenoviralvector DNA. As described herein, expression of a variantzymogen/protease polypeptide following administration of such anadenoviral vector serves to modulate hemostasis. In the context of thevariant Factor Xa described herein, such administration enhances theprocoagulation activity of the protease.

Recombinant adenoviral vectors have found broad utility for a variety ofgene therapy applications. Their utility for such applications is duelargely to the high efficiency of in vivo gene transfer achieved in avariety of organ contexts.

Adenoviral particles may be used to advantage as vehicles for adequategene delivery. Such virions possess a number of desirable features forsuch applications, including: structural features related to being adouble stranded DNA nonenveloped virus and biological features such as atropism for the human respiratory system and gastrointestinal tract.Moreover, adenoviruses are known to infect a wide variety of cell typesin vivo and in vitro by receptor-mediated endocytosis. Attesting to theoverall safety of adenoviral vectors, infection with adenovirus leads toa minimal disease state in humans comprising mild flu-like symptoms.

Due to their large size (˜36 kilobases), adenoviral genomes are wellsuited for use as gene therapy vehicles because they can accommodate theinsertion of foreign DNA following the removal of adenoviral genesessential for replication and nonessential regions. Such substitutionsrender the viral vector impaired with regard to replicative functionsand infectivity. Of note, adenoviruses have been used as vectors forgene therapy and for expression of heterologous genes.

For a more detailed discussion of the use of adenovirus vectors utilizedfor gene therapy, see Berkner, 1988, Biotechniques 6:616-629 andTrapnell, 1993, Advanced Drug Delivery Reviews 12:185-199.

It is desirable to introduce a vector that can provide, for example,multiple copies of a desired gene and hence greater amounts of theproduct of that gene. Improved adenoviral vectors and methods forproducing these vectors have been described in detail in a number ofreferences, patents, and patent applications, including: Mitani and Kubo(2002, Curr Gene Ther. 2(2):135-44); Olmsted-Davis et al. (2002, HumGene Ther. 13(11):1337-47); Reynolds et al. (2001, Nat Biotechnol.19(9):838-42); U.S. Pat. No. 5,998,205 (wherein tumor-specificreplicating vectors comprising multiple DNA copies are provided); U.S.Pat. No. 6,228,646 (wherein helper-free, totally defective adenovirusvectors are described); U.S. Pat. No. 6,093,699 (wherein vectors andmethods for gene therapy are provided); U.S. Pat. No. 6,100,242 (whereina transgene-inserted replication defective adenovirus vector was usedeffectively in in vivo gene therapy of peripheral vascular disease andheart disease); and International Patent Application Nos. WO 94/17810and WO 94/23744.

For some applications, an expression construct may further compriseregulatory elements which serve to drive expression in a particular cellor tissue type. Such regulatory elements are known to those of skill inthe art and discussed in depth in Sambrook et al. (1989) and Ausubel etal. (1992). The incorporation of tissue specific regulatory elements inthe expression constructs of the present invention provides for at leastpartial tissue tropism for the expression of the variantzymogen/proteases or functional fragments thereof. For example, an E1deleted type 5 adenoviral vector comprising nucleic acid sequencesencoding variant zymogen/protease under the control of a cytomegalovirus(CMV) promoter may be used to advantage in the methods of the presentinvention.

Exemplary Methods for Producing Adenoviral Vectors

Adenoviral vectors for recombinant gene expression have been produced inthe human embryonic kidney cell line 293 (Graham et al., 1977, J. Gen.Virol. 36:59-72). This cell line is permissive for growth of adenovirus2 (Ad2) and adenovirus 5 mutants defective in E1 functions because itcomprises the left end of the adenovirus 5 genome and, therefore,expresses E1 proteins. E1 genes integrated into the cellular genome of293 cells are expressed at levels which facilitate the use of thesecells as an expression system in which to amplify viral vectors fromwhich these genes have been deleted. 293 cells have been usedextensively for the isolation and propagation of E1 mutants, forhelper-independent cloning, and for expression of adenovirus vectors.Expression systems such as the 293 cell line, therefore, provideessential viral functions in trans and thereby enable propagation ofviral vectors in which exogenous nucleic acid sequences have beensubstituted for E1 genes. See Young et al. in The Adenoviruses,Ginsberg, ed., Plenum Press, New York and London (1984), pp. 125-172.

Other expression systems well suited to the propagation of adenoviralvectors are known to those of skill in the art (e.g., HeLa cells) andhave been reviewed elsewhere.

Also included in the present invention is a method for modulatinghemostasis comprising providing cells of an individual with a nucleicacid delivery vehicle encoding a variant zymogen/protease polypeptideand allowing the cells to grow under conditions wherein thezymogen/protease polypeptide is expressed.

From the foregoing discussion, it can be seen that zymogen/proteasepolypeptides, and zymogen/protease polypeptide expressing nucleic acidvectors may be used in the treatment of disorders associated withaberrant blood coagulation.

C. Pharmaceutical Compositions

The expression vectors of the present invention may be incorporated intopharmaceutical compositions that may be delivered to a subject, so as toallow production of a biologically active protein (e.g., a variantzymogen/protease polypeptide or functional fragment or derivativethereof). In a particular embodiment of the present invention,pharmaceutical compositions comprising sufficient genetic material toenable a recipient to produce a therapeutically effective amount of avariant zymogen/protease polypeptide can influence hemostasis in thesubject. Alternatively, as discussed above, an effective amount of thevariant Factor X polypeptide may be directly infused into a patient inneed thereof. The compositions may be administered alone or incombination with at least one other agent, such as a stabilizingcompound, which may be administered in any sterile, biocompatiblepharmaceutical carrier, including, but not limited to, saline, bufferedsaline, dextrose, and water. The compositions may be administered to apatient alone, or in combination with other agents (e.g., co-factors)which influence hemostasis.

In preferred embodiments, the pharmaceutical compositions also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce an immune responseharmful to the individual receiving the composition, and which may beadministered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, liquids such as water,saline, glycerol, sugars and ethanol. Pharmaceutically acceptable saltscan also be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., 18th Edition, Easton, Pa.[1990]).

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding, free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they may be placedin an appropriate container and labeled for treatment. Foradministration of zymogen/protease-containing vectors or polypeptides,such labeling would include amount, frequency, and method ofadministration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended therapeutic purpose.Determining a therapeutically effective dose is well within thecapability of a skilled medical practitioner using the techniques andguidance provided in the present invention. Therapeutic doses willdepend on, among other factors, the age and general condition of thesubject, the severity of the aberrant blood coagulation phenotype, andthe strength of the control sequences regulating the expression levelsof the variant zymogen/protease polypeptide. Thus, a therapeuticallyeffective amount in humans will fall in a relatively broad range thatmay be determined by a medical practitioner based on the response of anindividual patient to vector-based zymogen/protease treatment.

D. Administration

The variant Factor X polypeptides, alone or in combination with otheragents may be directly infused into a patient in an appropriatebiological carrier as described hereinabove. Expression vectors of thepresent invention comprising nucleic acid sequences encoding variantzymogen/protease, or functional fragments thereof, may be administeredto a patient by a variety of means (see below) to achieve and maintain aprophylactically and/or therapeutically effective level of thezymogen/protease polypeptide. One of skill in the art could readilydetermine specific protocols for using the zymogen/protease encodingexpression vectors of the present invention for the therapeutictreatment of a particular patient. Protocols for the generation ofadenoviral vectors and administration to patients have been described inU.S. Pat. Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; andInternational Patent Application Nos. WO 94/17810 and WO 94/23744, whichare incorporated herein by reference in their entirety.

Variant zymogen/protease encoding adenoviral vectors of the presentinvention may be administered to a patient by any means known. Directdelivery of the pharmaceutical compositions in vivo may generally beaccomplished via injection using a conventional syringe, although otherdelivery methods such as convection-enhanced delivery are envisioned(See e.g., U.S. Pat. No. 5,720,720). In this regard, the compositionsmay be delivered subcutaneously, epidermally, intradermally,intrathecally, intraorbitally, intramucosally, intraperitoneally,intravenously, intraarterially, orally, intrahepatically orintramuscularly. Other modes of administration include oral andpulmonary administration, suppositories, and transdermal applications. Aclinician specializing in the treatment of patients with bloodcoagulation disorders may determine the optimal route for administrationof the adenoviral vectors comprising zymogen/protease nucleic acidsequences based on a number of criteria, including, but not limited to:the condition of the patient and the purpose of the treatment (e.g.,enhanced or reduced blood coagulation).

The present invention also encompasses AAV vectors comprising a nucleicacid sequence encoding a variant zymogen/protease polypeptide.

Also provided are lentivirus or pseudo-typed lentivirus vectorscomprising a nucleic acid sequence encoding a variant zymogen/proteasepolypeptide

Also encompassed are naked plasmid or expression vectors comprising anucleic acid sequence encoding a variant zymogen/protease polypeptide.

Example 1 Variant Factor Xa Zymogen/Protease

Proteolytic processing of precursor plasma proteins to affect activationis a hallmark of blood coagulation. The paradigm for this type ofactivation mechanism is the zymogen to protease transition in thechymotrypsin-like serine protease family. Bond cleavage at a highlyconserved site (Arg15-Ile16; chymotrypsin numbering system) unmasks anew N-terminus which acts as an intramolecular ligand for Asp194 (FIG.2). This new salt-bridge results in or is associated with aconformational change and ordering of the so-called “activation domain”,surface loops consisting of the S1 specificity pocket, oxyanion hole,autolysis loop, and sodium biding site (FIG. 3). It is well documentedin the trypsin system that Ile16-Asp194 internal salt-bridge formationis allosterically linked to the S1 specificity site; that is changes atone site influence the other site and vice versa.

The basic principles of the zymogen to active enzyme transition forserine proteases at the structural level are well documented,particularly for chymotrypsin and trypsin and these examples serve asthe paradigm for the serine protease family. General aspects can besummarized as follows (see FIG. 2): 1) the structure (˜80-85%) of thezymogen is relatively similar to the protease; 2) the transition isinitiated following liberation of a highly conserved N-terminus (forexample, Ile16-Val-Gly-Gly19 (SEQ ID NO: 4)); 3) the new free α-aminogroup (Ile16) becomes buried in a hydrophobic environment and itsα-amino nitrogen forms an internal salt bridge with Asp194; 4) theposition of Asp194 changes significantly upon zymogen activationrotating ˜170°; and 5) this internal salt bridge results in or isassociated with a conformational change in the so-called “activationdomain”, surface exposed loops consisting of residues 16-19, 142-153,184-194, and 216-223; and partially comprising the S1 specificity site(nomenclature of Schechter and Berger (1967) Bochem. Biophys. Res. Comm.43:694-702) and oxyanion hole. Various studies indicate that the zymogenand the mature enzyme exist in an equilibrium, with Keq=˜10⁸ in favor ofthe zymogen. Bode and colleagues have elegantly demonstrated thattrypsinogen can adopt an active trypsin-like structure upon strongligand binding to the S1 specificity pocket or suitable ligands withhigh affinity for the Ile16 cleft. Additional examples of this inductionwithout cleavage of the Arg/Lys15-Ile16 bond include the binding ofstreptokinase to plasminogen, staphylocoagulase to prothrombin, and arecently described autoantibody to prothrombin (Madoiwa et al. (2001)Blood 97:3783-3789). Collectively these studies indicate that serineproteases, even in their zymogen forms, can adopt protease-likefunctions depending upon various environmental conditions, i.e. strongligand binding to the zymogen.

It is well known that the activation of FX results in majorconformational changes in the serine protease domain which areaccompanied by the ability of the protease to bind with much greateraffinity to S1-directed probes and membrane-bound FVa (1-6). The overallmolecular mechanism(s) which governs the transition of serine proteasesis generally assumed to follow that of the trypsin system. However, thismay not uniformly be the case. Single-chain tPA employs a differentmolecular strategy to maintain its zymogen-like state (8-11). Analysisof zymogen/protease pairs involved in blood coagulation, particularlyFVII/FVIIa, indicates that several differences exist in this transitioncompared to the trypsin system (12). While several structuraldeterminants on FXa are part of the so-called activation domain, it iscurrently unclear if formation of these sites is directly linked to thezymogen to protease transition. A recently described model of thezymogen FX suggests however that several of these elements may bedisordered in the zymogen (13). Comparison of the zymogen model with theactive enzyme reveals that residues making up the Ca²⁺ (Asp70-Glu80),Na⁺ (Ala183-Asp194; Gly219-Gly226) and autolysis loops (Thr144-Arg150)undergo major changes in their backbone positions upon the zymogen toprotease transition. Since it is already well-documented, at least fortrypsinogen/trypsin, that the S1 specificity site and formation ofIle16-Asp194 are allosterically linked, it is reasonable to hypothesizethat other elements of the activation domain are also linked to thezymogen to protease transition. In the present example, we have designedexperiments to test the hypothesis that destabilization of theIle16-Asp194 internal salt bridge by making changes to position 16, 17,or 194 alters the active site cleft making the resulting variant“zymogen-like”. We also hypothesized that these changes wouldallosterically modulate FVa binding.

Materials and Methods

Expression of Factor Xa

While there are several reports in the literature on the expression ofrFX, most have relied on truncated versions or have not providedadequate characterization (15-20). Our initial attempts at expressingrFX in HEK 293 cells resulted in expression levels in the range of 1-2mg rFX/L of conditioned media; however, only 10-40% of the materialproduced was found to be fully γ-carboxylated (21). The remainingmaterial showed no γ-carboxylation. We took advantage of the differentbinding affinities of the vitamin K-dependent propeptides for thecarboxylase and hypothesized that since the prothrombin propeptideexhibits the lowest affinity for the carboxylase, exchanging thepropeptide of FX (highest affinity) with that of prothrombin may enhanceγ-carboxylation by allowing for greater substrate turnover (22, 23).Using this new vector, stable transfectants of HEK 293 cells wereselected, expanded, and rFX was purified by immunoaffinitychromatography. Phosphate elution from hydroxyapatite was used toseparate carboxylated material away from uncarboxylated material. Ourresults, obtained now from over 30 stable cell lines indicate that onaverage ˜80-90% of the rFX is fully γ-carboxylated compared to 10-40%using the native FX propeptide. These results have recently beenpublished and this strategy has subsequently been employed by at leastone other laboratory (24,25). Thus, using this new expression system weare now producing milligram quantities (15-25 mg of fully γ-carboxylatedrFX from ˜10 L of conditioned media) of protein for detailedstructure/function studies.

Enzyme Assays

Enzyme concentrations will be determined by active-site titration withρ-nitrophenyl ρ-guanidinobenzoate (IIa) or fluoresceinmono-ρ-guanidinobenzoate (FXa) (26, 27). FXa chromogenic substrateactivity, in the presence or absence of various inhibitors, will bemeasured from initial rates of hydrolysis of Spectrozyme FXa, S-2222, orS-2765 as previously described (14). Kinetic parameters will bedetermined by least-squares fitting of the initial rate data toappropriate equations.

Generation of FXa Variants

The FX mutants were generated using the Quick-change site-directedmutagenesis kit (Stratagene) and the entire FX cDNA was sequenced toverify the identity of the product. The various plasmids weretransiently transfected into HEK 293 cells using Lipofectamine-2000. 48hr post-transfection, media was collected and FX antigen levels weredetermine using a FX-specific ELISA and FXa activity was assessed bychromogenic assay prior to activation by RVV-X or by tissuefactor-FVIIa.

Results

Generation of FXa species distributed along the zymogen-proteasetransition pathway is outlined in Table 1. Transient transfectionresults indicate that we have generated a series of FXa variants withvariable amounts of activity, ranging from ˜25% to <1%. We hypothesizethat these differences in activity likely reflect FX variants which areshifted to varying degrees along the zymogen to protease transition.Stated differently, the Ile16-Asp194 internal salt bridge is stabilizedto varying degrees depending upon the amino acid at positions 16, 17 or194. We have chosen three of these variants (rFXaI16L, FXaI16G andFXaV17A) for further characterization.

TABLE 1 Activation of Various rFX Variants with RVV-X and TF-FVIIaActivation of Activation of FX with RVV-X: FX with TF-FVIIa: Constructs^(a)[FX] (nM) % wt-Antigen ^(b)FXa Activity (nM) ^(c)% wt ^(b)FXaActivity (nM) ^(c)% wt rwt-FX 54.07 100.00 10.95 100.00 18.905 100.00Ile16→Leu 24.88 46.03 0.25 5.00 0.572 6.57 Ile16→Phe 49.48 91.51 0.010.08 0.004 0.02 Ile16→Asp 12.04 22.27 0.01 0.22 0.000 0.00 Ile16→Gly27.88 51.56 0.00 0.05 0.037 0.38 Val17→Leu 28.18 52.12 1.22 21.33 3.00330.48 Val17→Ala 55.88 103.36 0.34 3.02 1.029 5.27 Val17→Gly 47.76 88.340.02 0.21 0.036 0.22 Asp194→Asn 17.32 32.04 0.03 0.79 0.000 0.00Asp194→Glu 30.97 57.29 0.02 0.27 0.000 0.00 ^(a)Antigen levels arecalculated from a FX-specific ELISA and expressed as nM FX ^(b)FXaactivity levels are based on the rate of peptidyl substrate hydrolysisfollowing activation by RVV-X or TF-FVIIa of a given FX variant andinitial rates of hydrolysis are compared to FXa standard curve. ^(c)% wtvalues are based upon comparison to wt-FXa activity levels. The valueshave been adjusted for antigen levels.

Stable cell lines in HEK293 cells were established and each of thezymogens were purified from 10 L of conditioned media (14, 24). Thevariants were activated with RVV-X and subsequently purified by gelfiltration chromatography (14,24). SDS-PAGE of the variants prior to andfollowing activation (reducing and non-reducing) is shown in FIG. 4.

We first focused on assessing changes to the active site environment ofeach of the variants using specific probes that target this region ofFXa. Kinetic studies using peptidyl substrates and active site directedprobes revealed that FXaI16L and FXaV17A have an impaired ability tobind these probes (15 to 25-fold increase in the Km or Ki) while therate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa(plasma-derived and recombinant) (Tables 2 and 3). Factor Xa I16G wasnot inhibited by any of the probes examined and its chromogenic activitywas severely impaired (500 to 1000-fold) precluding calculation ofkinetic parameters. These data are consistent with the idea thatdestabilization of internal salt-bridge formation (Ile16-Asp194),influences binding at the S1 specificity site. In contrast to theseresults, the assembly of FXaI16L and FXaV17A into prothrombinase almostcompletely restored the Km for peptidyl substrates while the kcat wasstill 3-fold reduced, indicating that FVa binding can rescue binding atthe active site (Tables 2 and 3). Surprisingly even the Km value for116G was almost completely restored (3-fold increased compared towild-type FXa) when assembled in prothrombinase; however a 60-foldreduction in the kcat was found.

TABLE 2 Kinetic constants for chromogenic substrate cleavage EnzymeSpecies K_(m) (μM) ± SD k_(cat) (sec⁻¹) ± SD Factor Xa rwtFXa 15X  88.8± 11.4 3X  215 ± 13.5 rFXa^(V17A) 1377 ± 332 71.6 ± 13.5 rFXa^(I16L)1149 ± 244 57.3 ± 3.1  rFXa^(I16G) 1608 ± 423 0.28 ± 0.05 ProthrombinaserwtFXa  3X 130 ± 11 3X 141 ± 3.7  rFXa^(V17A) 362 ± 42 68.2 ± 9.7 rFXa^(I16L) 296 ± 54 32.5 ± 3.1  rFXa^(I16G) 433 ± 31 60X  1.92 ± 0.05For experiments in which free factor Xa was used, 2.0 nM wild-type or6.0 nM mutant factor Xa was incubated with increasing concentrations ofSpectrozyme FXa and for experiments in which prothrombinase was employed5.0 nM wild-type or mutant factor Xa was incubated with 30 nM factor Va,50 μM PCPS and increasing concentrations of substrate (10 to 500 μM).Chromogenic activity was assessed by monitoring the increase inabsorbance at 405 nm over time. The errors in the fitted constantsrepresent 95% confidence limits.Consistent with these data, kinetic studies using prothrombin revealedthat the Km values obtained for each of these variants assembled inprothrombinase were essentially equivalent to the wild-type enzyme,while the kcat values where reduced to a similar extent as for thechromogenic substrates (Table 4). Taken together, our results indicatethat the zymogen to protease transition for FX not only influences theformation of the S1 site, but also contributes in a substantial way tothe formation of a FVa binding site. Since direct binding of these FXavariants to FVa rescues binding at S1 site, allosteric linkage likelyexists between these sites. Collectively these studies have illustrateda unique way to modify the zymogen to protease transition pathway andhave revealed a possible way to develop zymogen-like forms of enzymeswhich are “activated” following strong ligand binding such as cofactorproteins.

TABLE 3 Kinetic constants for inhibition of FXa and prothrombinase K_(i)(μM) ± SD k₂ (M⁻¹ s⁻¹) ± SD × 10³ Enzyme Species Pefabloc tPa/XaPara-amino benzamidine Antithrombin III Factor Xa rwtFXa 25X 0.070 ±0.002 11X 78.1 ± 1.5 19X 1.37 ± 0.02  rFXa^(V17A) 1.695 ± 0.072 996 ± 370.09 ± 0.003 rFXa^(I16L) 1.701 ± 0.065 726 ± 40 0.06 ± 0.001Prothrombinase rwtFXa  5X 0.050 ± 0.002  3X 48.6 ± 0.6  4X 0.28 ± 0.01 rFXa^(V17A) 0.295 ± 0.013  191 ± 6.4 0.05 ± 0.001 rFXa^(I16L) 0.209 ±0.005  143 ± 9.0 0.09 ± 0.003

TABLE 4 Kinetic constants for prothrombin cleavage K_(m) ± SDV_(max)/E_(t) ± SD V_(max)/E_(t) · K_(m) Enzyme species (μM) (nMIIa/min/nM E) (μM⁻¹ · s⁻¹) pdFXa 0.42 ± 0.02 2424 ± 54 3X 96 rFXa 0.35 ±0.01 1937 ± 26 92 FXa^(V17A) 0.47 ± 0.03  887 ± 26 31 FXa^(I16L) 0.31 ±0.02  619 ± 14 33

The results obtained with the chromogenic substrate and the activesite-directed inhibitors, indicate that the zymogen-like FXa variantsbind to active site probes with reduced affinity. However, assembly ofthese variants into prothrombinase significantly improves the affinityfor active site probes, suggesting that FVa binding can rescue bindingat the active site. We next investigated whether the reverse is alsotrue, that is occupation of the zymogen-like active site influencesbinding to FVa. In order to assess this hypothesis we measured thebinding constants between FVa and FXaI16L and FXaV17A. To accomplishthis we incubated FVa with a fluorescent derivative of inactive wtFXa inthe presence of synthetic phospholipid vesicles and Ca2+ ions. Theformation of the complex results in the increase of the fluorescentsignal relative to fluorescent FXa alone. We then added increasingconcentrations of a non-fluorescent FXa which, if it can bind FVa, willdisplace the fluorescent FXa, resulting in a decrease of the fluorescentsignal. As a control we added S195A FXa as a competitor. This mutant isinactive because it is missing the Ser of the catalytic triad, but has ahigh affinity for FVa (Table 5). In contrast, when we added eitherFXaI16L or FXaV17A the affinity of these zymogen-like variants for FXawas significantly reduced compared to FXaS195A (Table 5). We nextexamined whether covalent occupation of the active site of FXaI16L couldrestore binding to FVa. To do this we modified the active site ofwild-type FXa and FXaI16L with an irreversible inhibitor(EGR-chloromethyl ketone) and then repeated the experiment. The datashow that that active site blocked FXaI16L bound FVa-membranes with thesame affinity as wild-type active site blocked FXa. This indicates thatoccupation of the zymogen-like FXa active site has a direct influence onthe binding to FVa.

TABLE 5 Equilibrium binding constants for prothrombinase assembly Enzymespecies K_(d) ± SD (nM) rFXa^(S195A) 1.34 ± 0.17 rFXa^(V17A) 7.25 ± 0.65rFXa^(I16L) 13.81 ± 1.07  EGR-FXa 1.80 ± 0.42 EGR-FXa^(I16L) 1.92 ± 0.20

Based on the observation that the zymogen-like FXa derivatives have poorreactivity with active-site directed probes and inhibitors in theabsence of FVa, but apparently near normal activity when the variantsare assembled in prothrombinase, we next evaluated the activity ofFXaI16L in a plasma environment. Hemophilic A (data not shown) or Bplasma was spiked with wild-type FXa to correct the clotting time (aPTT)of these plasmas; 0.1 nM wtFXa gave a clotting time of ˜32 sec. Theaddition of the same concentration of FXaI16L gave a clot time of ˜42sec which is ˜50-70% of the activity relative to wtFXa, suggesting thatthis zymogen-like variant has almost normal clotting activity in plasma.Next we monitored the half-life of wild-type FXa and FXaI16L inhemophilia B plasma. The proteins were added to HB plasma and atdifferent time points, an aliquot of the mixture was withdrawn andassayed in an aPTT-based assay. The results with HB plasma show that therelative residual activity of wild-type FXa was inhibited very rapidly(<2-min) (FIG. 6). In contrast, the activity of FXaI16L persisted for amuch longer time with an estimated half-life of >2 hours. Similarresults were found with hemophilia A plasma. These results suggest thatit is possible to modulate the characteristics of an enzyme so that ithas a long half-life in plasma and can correct the clotting time of ahemophilic plasma.

We next evaluated the ability of zymogen-like FXaI16L to modulatehemostasis in a murine model of hemophilia (Schlachterman, et. al.,2005, J. Thromb. Haemost., 3, 2730-2737). The aPTT value of hemophilia Bmice (C57BL/6) is approximately 50-55 sec. Factor XaI16L (200 μg/kg;n=7) or PBS (n=4) were injected via the tail vein of hemophilia B mice.At selected time points (5 and 30 min) blood was collected and an aPTTwas performed on all samples. As shown in FIG. 7, infusion of FXaI16Lresulted in complete correction of the aPTT to levels seen in normalanimals. This effect was sustained for at least 30 min indicating thatthe molecule has a relatively long half life in vivo. Infusion of PBShad only a marginal effect. These data are consistent with the in vitroplasma experiments above and indicate that indeed FXaI16L and possiblyother zymogen-like FXa variants can effectively modulate hemostasis invivo.

To further test the effectiveness of FXaI16L in vivo, we examinedwhether this molecule could correct the bleeding time of hemophilia Bmice following injury to the tail (Schlachterman, et. al., 2005, J.Thromb. Haemost., 3, 2730-2737). Blood loss was measured during a 10-minperiod after sectioning the distal part of the tail. In this type ofassay, blood loss is minimal in normal wild-type Balb-c mice (n=7) andquite substantial in PBS injected (n=6) hemophilia B mice (Balb c)following the tail injury (FIG. 8). In contrast, injection of 450 μg/kgof FXaI16L significantly reduced the total amount of blood lossfollowing tail injury (n=7). Taken together these data provide evidencethat FXaI16L has the ability to improve hemostasis in hemophilia A or Bpatients.

REFERENCES

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A nucleic acid molecule comprising a nucleic acidsequence encoding a two chain human Factor Xa variant protein comprisinga light chain and a heavy chain, wherein the amino acid at the aminoterminus of the heavy chain, at the position corresponding to amino acidnumber 235 in SEQ ID NO:1, is Leucine.
 2. A nucleic acid moleculecomprising a nucleic acid sequence encoding a human Factor Xa variantprotein comprising a light chain and a heavy chain, wherein the lightchain comprises amino acid numbers 41 to 179 of SEQ ID NO:1; and theheavy chain comprises Leucine at its amino terminus followedcontiguously by amino acid numbers 236 to 488 of SEQ ID NO:1.
 3. Thenucleic acid molecule of claim 2, wherein the nucleic acid sequenceencoding the light chain of said human Factor Xa variant proteincomprises nucleotide numbers 121 to 537 of SEQ ID NO:2; and the nucleicacid sequence encoding the heavy chain of said human Factor Xa variantprotein comprises a codon encoding Leucine followed contiguously bynucleotide numbers 706 to 1464 of SEQ ID NO:2.
 4. A nucleic acidmolecule comprising a nucleic acid sequence encoding a human Factor Xvariant protein comprising a light chain sequence comprising amino acidnumbers 41 to 179 of SEQ ID NO:1, the amino acid sequenceArginine-Lysine-Arginine, corresponding to amino acid numbers 180 to 182of SEQ ID NO:1, an Activation Peptide sequence corresponding to aminoacid numbers 183 to 234 of SEQ ID NO:1; and a heavy chain sequencecomprising at Leucine its amino terminus followed contiguously by aminoacid numbers 236 to 488 of SEQ ID NO:1.
 5. The nucleic acid molecule ofclaim 4, wherein the nucleic acid sequence encoding the ActivationPeptide sequence is substituted with a nucleic acid sequence encoding anintracellular protease cleavage site.
 6. The nucleic acid molecule ofclaim 5, wherein the intracellular protease cleavage site is recognizedand cleaved by a PACE or furin enzyme.
 7. The nucleic acid molecule ofclaim 4, wherein the nucleic acid sequence encoding the ActivationPeptide sequence is substituted with a nucleic acid sequence encodingthe amino acid sequence Arginine-Lysine-Arginine.
 8. The nucleic acidmolecule of claim 4 further comprising a nucleic acid sequence encodinga propeptide.
 9. The nucleic acid molecule of claim 8, wherein thepropeptide is the thrombin propeptide.
 10. The nucleic acid molecule ofclaim 7 further comprising a nucleic acid sequence encoding a signalpeptide.
 11. The nucleic acid molecule of claim 4, wherein said nucleicacid sequence encoding a human Factor X variant protein comprisesnucleotide numbers 121 to 1464 of SEQ ID NO:2, wherein the codonencoding Isoleucine at nucleotide numbers 703 to 705 of SEQ ID NO:2 issubstituted with a codon encoding Leucine.
 12. The nucleic acid moleculeof claim 11, wherein nucleotide numbers 547 to 702 of SEQ ID NO:2,encoding the Activation Peptide, are substituted with codons encodingthe amino acid sequence Arginine-Lysine-Arginine.
 13. A nucleic acidmolecule comprising a nucleic acid sequence encoding a human Factor Xvariant protein comprising, in order, a thrombin propeptide, amino acidnumbers 41 to 182 of SEQ ID NO:1, the amino acidsArginine-Lysine-Arginine, the amino acid Leucine; and amino acid numbers236 to 488 of SEQ ID NO:1.
 14. The nucleic acid molecule of claim 13,wherein amino acid numbers 41 to 182 of SEQ ID NO:1 are encoded bynucleotide numbers 121 to 546 of SEQ ID NO:2; and amino acid numbers 236to 488 of SEQ ID NO:1 are encoded by nucleotide numbers 706 to 1464 ofSEQ ID NO:2.
 15. An expression vector comprising the nucleic acidmolecule of claim 1 operably linked to a transcription control element.16. An expression vector comprising the nucleic acid molecule of claim 2operably linked to a transcription control element.
 17. An expressionvector comprising the nucleic acid molecule of claim 3 operably linkedto a transcription control element.
 18. An expression vector comprisingthe nucleic acid molecule of claim 7 operably linked to a transcriptioncontrol element.
 19. An expression vector comprising the nucleic acidmolecule of claim 13 operably linked to a transcription control element.20. An expression vector comprising the nucleic acid molecule of claim14 operably linked to a transcription control element.
 21. An expressionvector comprising the nucleic acid molecule of claim 2 selected from thegroup consisting of adenoviral vectors, lentivirus vectors, felineimmunodeficiency virus (FIV) vectors, herpes simplex virus vectors,vaccinia virus vectors, and retroviral vectors.
 22. The vector of claim21 which is an adeno-associated virus (AAV) vector.
 23. The vector ofclaim 22, selected from the group consisting of AAV-2, AAV-5, AAV-7, andAAV-8 and hybrid AAV vectors.